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Fig. 2. Subcellular localization of Prep1.1-GFP. (A) Full-length zebrafish prep1.1, as sequenced from the EST fc13f10, is represented with the Meis family homology regions (HR1 and HR2) in blue and the homeodomain homology region (HD) in red. The positions of the morpholinos (MOs) relative to the cDNA sequence are indicated by orange bars. prep1.1 constructs, with GFP coding sequence in green, are also shown. prep1.1-MOb inactivates endogenous, full-length mRNA but not the full-coding constructs. (B-J) Subcellular localization of Prep1-1-GFP chimeric proteins. (B-D,H) In a wild-type embryo injected with 10 pg of prep1.1-GFP mRNA, Prep1.1-GFP, which is cytoplasmic at the high stage (not shown), is translocated to the nucleus from the sphere stage onwards. Starting from the shield stage, the fluorescent protein is restricted mostly to the nucleus. (E-G) In a pbx4 morphant injected with 6 ng pbx4-MO and 10 pg prep1.1-GFP mRNA, Prep1.1-GFP is also translocated to the nucleus from the sphere stage onwards, but significant amounts are detected in the cytoplasm in all subsequent stages. (I,J) In wild-type embryos, Prep1.1HD-GFP, a derivative lacking the homeodomain, is translocated normally to the nucleus (I), whereas Prep1.1HR-GFP, a derivative lacking the Meis homology region is not (J). (B-D,H) and (E-G) are from the same wild-type and pbx4 morphant embryos, respectively. (K) Western blot of extracts from 24 hpf zebrafish embryos. The left panel is a control blot to assess the migration of two Pbx proteins, mPbx1a and mPbx1b, of known molecular weight and antibody specificity. The two proteins, which are recognized specifically by the pan-Pbx antibody, were translated in vitro with hPrep1 in a rabbit reticulocyte system. The middle panel is another control using nuclear extracts (NE) and cytoplasmic extracts (CE) of either human HEK293 cells or mouse testis. The right panel shows the immunoblotting analysis of NE and CE from wild-type zebrafish embryos at 24 hpf and following injection of prep1.1-MOb. The migration of Pbx2 and Pbx4 (arrows) is inferred on the basis of the M_r of the bands, and observation of the identical pattern elsewhere (Waskiewicz et al., 2002). A pan-Pbx antibody (Pöpperl et al., 2000) was used throughout.

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